Regulatory glycoprotein for immune response and the use, thereof in the production of t-cell growth factor

ABSTRACT

An interleukin-1 preparation is obtained as a supernatant by cell culturing human peripheral mononuclear blood cells.

This application is a continuation of application Ser. No. 07/051,199,filed May 18, 1987, now abandoned, which is a continuation of Ser. No.06/730,045, filed May 3, 1985, now abandoned, which is a division ofapplication Ser. No. 06/514,197, filed July 15, 1983, now U.S. Pat. No.4,661,447, which in turn is a division of Ser. No. 06/255,251, filedApr. 17, 1981, now U.S. Pat. No. 4,406,830.

BACKGROUND OF THE INVENTION

This invention relates to a newly discovered blood serum protein whichis part of the T-cell regulating system; to the use of this novelprotein in the production of a serum-free and mitogen-free preparationcontaining Interleukin-I and a serum-free and mitogen-free preparationcontaining T-cell growth factor; to a chemically defined T-cell growthculture medium containing the novel protein as the only protein; and tothe novel protein in a purified form.

A serum-free, mitogen-free T-cell growth factor (Interleukin-2 or Il-2)has been described in the pending application of Hans-Ake Fabricius andRoland Stahn, titled A SERUM-FREE AND MITOGEN-FREE T-CELL GROWTH FACTORAND PROCESS FOR MAKING SAME, Ser. No. 247,769, filed Mar. 24, 1981 nowU.S. Pat. No. 4,390,623 (filed as a continuation-in-part of applicationSer. No. 193,112, filed Oct. 2, 1980, now abandoned). The disclosures ofthese prior applications are incorporated herein, in their entirety byreference thereto.

The preparation of the T-cell growth factor described in these priorapplications includes a step of stimulating isolated lymphocytes (e.g.peripheral mononuclear blood cells) in the presence of serum and amitogen such as phytohemagglutinin A (PHA). In fact, all known publishedliterature on the production of lymphokines from lymphocytes reports thestep of contacting the lymphocytes with mitogen in the presence of bloodserum, added as a supplement to the culture medium.

While such procedures produce Il-2 in significant yields the presence ofserum has the drawbacks referred to in the above-mentioned priorapplications, namely the presence of numerous proteins which may maskthe true effect of Il-2; allergic and anaphylactic reactions in patientsrepeatedly given injections of serum-containing preparations; and theinability to concentrate serum-containing preparations since the serumwill precipitate and plug the pores of the filters used for thispurpose. Despite these drawbacks it had always been presumed that thepresence of serum was essential for the production and maintenance ofgrowing T-cell lines by the T-cell growth factor (Il-2). However, theinventors of the above mentioned prior applications have been able toshow that serum-free and nitrogen-free Il-2 preparations can be used tomaintain growing T-cell lines, and thereby provide a means forcombatting tumor growth.

Nevertheless, the procedure described in the prior applications whichrequired the lymphocyte stimulating step in the presence of serum wassomewhat cumbersome in requiring the essentially complete removal of theserum prior to incubating the stimulated cells in a serum-free,mitogen-free liquid culture medium.

The present inventors conceived the idea that it might be possible tocompletely eliminate the use of serum in the stimulation of thelymphocytes. In order to test this idea it was first attempted to testthe effect of specific fractions of human serum which had been separatedin narrow molecular weight ranges by gel partition chromatography. Quitesurprisingly it was found that only the molecular weight fraction of theserum in the range of from about 60,000 to 130,000 was capable ofinducing production of Il-2. However, none of the known blood serumproteins in this molecular weight range, e.g. transferrin, haptoglobin,albumin, etc. were found to be capable of inducing the production ofIl-2 in bioassay tests in culture medium. This observation led theinventors to the conclusion that an unidentified blood serum factor mustbe present in the serum and must be necessary for the stimulation oflymphocytes to produce T-cell growth factor (also known as TCGF orIl-2). In fact, such previously unidentified and unknown blood serumfactor has now been isolated and provides the basis of the presentinvention.

SUMMARY OF THE INVENTION

According to the present invention, a previously unknown unidentifiedblood serum protein (glycoprotein) having a molecular weight of about90,000 Daltons and having at least one sialic acid (N-acetyl neuraminicacid) residue is provided. This protein is further characterized by itsrelative heat stability - it loses less than one-half of its biologicalactivity when heated at 80° C. for 30 minutes; and by its strongaffinity to lectins, primarily wheat germ agglutinin (WGA), and tohydrophobically binding ligands such as Blue A, which are commerciallyreadily available; but no specific binding to lysine.

In another aspect this invention provides a process for producingserum-free, mitogen-free Interleukin-1 (Il-1) (also known as lymphocyteactivating factor LAF) and serum-free, mitogen-free Il-2 by incubatingperipheral mononuclear blood (PBL) cells in a serum-free liquid tissueculture medium to remove residual serum proteins on the surfaces of thePBL cells, activating the incubated cells with a mitogen, washing theactivated cells with a sterile liquid to remove the mitogen from thecells and conditioning the serum-free mitogen-free activated cells in aliquid tissue culture medium to produce a serum-free, mitogen-freeInterleukin-1 (Il-1); contacting the Il-1 containing liquid tissueculture medium with the novel blood serum glycoprotein of thisinvention; and incubating the cells in the presence of Il-1 and thenovel blood serum glycoprotein to induce synthesis of Il-2 and totransfer the Il-2 (T-cell growth factor) from the cells to the liquidphase of the tissue culture medium to thereby produce a serum-free,mitogen-free Il-2.

In still another aspect this invention provides a chemically-definedliquid tissue culture growth medium for growing lymphocytes in vitro.The medium includes the conventional cell nutrients and as the onlyprotein constituent of the medium the novel blood serum protein of thisinvention. This tissue culture growth medium can then be used to growT-cells in the absence of blood serum.

Such a medium is beneficial for studying regulation of T-lymphocytegrowth in a chemically defined surrounding, and offers the advantage ofbeing less dangerous with respect to hepatitis than media containinghuman whole serum.

A still further aspect of this invention relates to a process forinhibiting the in vivo production of natural killer cells by interferingwith the cell mediated immunological system response mechanism toinvasion by foreign bodies, particulary grafts of tissue or otherorgans. According to this process a serine alkylating agent is used toprevent blastogenic transformation of T-cells to T-blasts.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be explained in greater detail by the followingdescription and specific embodiments and with the aid of theaccompanying drawings in which:

FIG. 1 is a plot of light transmission (%) versus elution volume forseparation of human blood serum on a Sephadex G 200 chromatographycolumn and also showing the biologically active peak for the novel bloodserum glycoprotein of this invention and also the peaks for transferrinand haptoglobin.

FIG. 2 is a plot of light transmission (%) versus elution volume for theseparation of the biologically active fractions (Nos. 75-100) obtainedin Example 1 after refractionation on a Sephadex G 150 chromatographycolumn.

FIG. 3 is a plot showing the results of chromato-focussing thebiologically active fractions (Nos. 75-100) obtained in Example 1 interms of biological and immunological activity; and

FIG. 4 is a graph showing the results obtained in Example 7 for theproduction of Interleukin 2 in the presence and in the absence of thenovel blood serum glycoprotein of this invention.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

It has been demonstrated in the literature and also shown in the datareported in the above-mentioned applications of Fabricius and Stahn thattumor patients are defective in the ability of their cellular immunesystem (cell mediated immunity) to produce the T-cell growth factor(Il-2) necessary for production of natural killer cells which can attackand destroy tumors. Therefore, if the natural killer cells in tumorpatients can be stimulated into activity, it should be possible to stopthe growth of tumor cells.

According to current theory of the cell mediated immunological responsemechanism to invasion of the host by a mitogen, the mitogen activates amacrophage subpopulation of lymphocytes to produce Il-1 (Interleukin I)which in turn stimulates a T-cell subpopulation, presumably T-helpercells, to produce Il-2. The latter is identified biologically by itsability to support the growth of permanently growing T-cell lines.

Contrary to this current belief it has now been found that production ofIl-2 not only depends on the interaction of T-lymphocytes with Il-1 butalso with a previously unknown unidentified protein found in bloodplasma sera from humans as well as from other mammals , such as horsesera and bovine sera. This protein has a molecular weight about 90,000.Thus, when Il-1 is brought into contact with lymphocytes in the absenceof serum or with serum from which the molecular weight fraction range of86,000 to 94,000 has been eliminated by fractionation Il-2 production isnot observed. As far as applicants are aware this observed phenomenonhas never been reported or suggested in the literature.

In order to determine whether this "comitogenic" blood serum factor wasone of the known blood serum proteins in the molecular weight rangewhere the biological activity was found a series of bioassay tests werecarried out by adding the known proteins- albumin, transferrin,plasminogen and haptoglobin to an Il-1 conditioned serum-free cellculture medium. None of these known proteins were capable of formingT-cell blasts or sustaining T-cell lines. Accordingly, it was concludedthat none of these proteins were capable of interacting with Il-1 on theT-cell lymphocytes to produce Il-2 (Il-2 by definition is capable ofsustaining the growth of T-cell lines).

The unknown "comitogenic" factor was determined to be a protein byvirtue of the fact that the biological activity of the "comitogenic"factor-containing blood serum fraction was destroyed by treatment withtrypsin. Since trypsin only deactivates proteins (by destroying thepeptide bonds) the unknown factor must be a protein.

Further investigation revealed the presence of at least one sialic acidresidue (N-acetyl neuraminic acid). Thus, treatment of the novelcomitogenic protein factor with neuraminadase enzyme resulted in a loseof biological activity.

By the reaction of the comitogenic protein factor with various lectinsit has been further identified as a glycoprotein although the specificsugar moiety has not yet been identified.

Since the comitogenic glycoprotein was originally found in human bloodsera it has been given the acronym PHILIP: Plasmatic Human InterleukinInducing Protein. Accordingly, the name PHILIP may be used to identifythe previous unknown and unrecognized blood serum protein which can becharacterised by its physico-chemical properties: molecular weight ofabout 90,000 D; glycoprotein; at least one sialic acid residue; strongadherence (binding) to lectins such as wheat germ agglutinin, and BlueA; and relative heat stability; and by its biological activity: in thepresence of Interleukin-I (serum-free and mitogen-free) will induceperipheral monuclear blood cells (lymphocytes: T-cells) to produceT-cell growth factor (IL-2) which forms T-cell blasts and supports thegrowth of T-cell lines; alternatively, in a serum-free system, in thepresence of mitogen, such as PHA and peripheral mononuclear blood cells,PHILIP will induce the production of Il-2 and T-cell blastogenesis. Inthe latter case the PHA first induces the pro-macrophage cells toproduce Il-1, which, with the coaction of PHILIP, induces a second cellfraction, presumably T-helper cells, to produce Il-2.

At this point it should be emphasized that the mechanism by which PHILIPfunctions to cooperate with Il-1 and the T-cells to produce Il-2 has notbeen elucidated. It is generally believed, but has not yet been provenconclusively, that the subpopulation of T-cells known as T-helper cellsproduce Il-2.

The production of an Il-1 preparation which is serum-free andmitogen-free is an important and novel aspect of this invention. SuchIl-1 preparation can be used directly, or after being concentrated(which is made possible by being serum-free) for production of Il-2.

The serum-free mitogen-free Il-1 can be prepared from isolated adherent(pro-macrophage) cells or directly from peripheral mononuclear bloodcells (PBL). To isolate the adherent (pro-macrophage) cells thefollowing well known technique can be used: Peripheral mononuclear bloodcells are first isolated from the blood by the density gradienttechnique. For example, the blood is layered into a tube containing ahigh density polymeric solution, such as the commercially availableFicoll-hypaque, and centrifuged. The PBL cells are collected above thepolymeric solution and are carefully removed. The Ficoll-hypaquesolution is slightly toxic so the removal must be done very carefully.The so-obtained PBL are washed serveral times, usually three times, witha sterile solution to ensure complete removal of the Ficoll-hypaquesolution. The washing is accomplished by gently suspending the cells inthe sterile solution and centrifuging to again separate the cells fromthe solution. Low centrifuge speed andtime (e.g. 300×G for ten minutes)are used to avoid breakage or destruction of the cells. Rosewell ParkMemorial Institute (RPMI) 1640 is a commercially available sterilesolution which can be used for this purpose and also as the liquidculture medium. Machines are presently available for the automaticseparation of mononuclear cells from blood and such machines areadvantageously used when available.

The PBL cells are then placed in plastic Petri dishes and incubated forabout 1 hour at about 37° C. The pro-macrophage cells are adherent toplastic and adhere to the walls of the Petri Dishes. The non-adherentcells are washed off by spraying with sterile solution, e.g. RPMI 1640.The adherent cells are removed from the Petri dish. A rubber policemancan conveniently be used for this purpose.

The recovered pro-macrophage cells, after being counted (usually about3×106 cells per ml are used) are incubated for from about 18 to 24 hoursat about 37° C. using a nonsupplemented liquid culture medium, such asRPMI-1640. Fresh culture medium can be added to or replace used culturemedium every few hours if desired. By "non-supplemented" is meant thatno mitogenic substances or blood serum is added to the culture. Allincubation steps in this invention are carried out under a CO₂ (about5-15%) atmosphere and a temperature of about 37° C., unless otherwisenoted.

The purpose of this preliminary incubation step is to remove any serumproteins which may have been present on the cell membrane surfaces.Since the cell membranes are regenerated about once every eight hoursthe membranes will "turn over" about 2 or 3 times during this incubationperiod.

When using the PBL cells without separation of the adherentpro-macrophage cells the PBL cells can be directly incubated under thesame conditions as described above to remove any proteins which may bepresent on the surface (membrane of the cells. The cells are now readyto be activated for production of Il-1.

Activation of the cells is accomplished by contacting the cleaned cellsin the liquid culture medium with a mitogen. Phytohemagglutinin A (PHA)is preferably used for this purpose although other lectin typemitogens,.such as concanavalin A (Con A), Escherichia colilipopolysaccharide (LPS) and poke-weed mitogen (PWM) can also be used.An important feature of the invention is that the mitogen is only leftin contact with the cells for a short period of time, preferably about 4hours although longer times up to about 12 hours can be used. Since thecells are activated or stimulated for and actually begin production ofIl-1 during this short time period the mitogen is removed from the cellswithout significantly limiting the production of Il-1.

The washing can be accomplished as described above for removing theFicoll-hypaque solution, i.e. centrifuging the cell suspension, addingthe cells to fresh sterile solution, and repeating this process about 3times. The supernatants can be saved for future cell stimulations or forrecovery of Il-1 or PHA.

The activated cells can then be conditioned by incubating the cells in afresh non-supplemented RPMI 1640 for about 12 to 30 hours, preferablyabout 15 to 26 hours, most preferably about 14-24 hours. Generally, thecells are used in a concentration of about 3×10⁶ cells per ml of culturemedium.

A serum-free lectin free Il-1 is obtained in the supernatant. Thepresence of Il-1 is confirmed by known chemical and biologicalcharacteristics of Interleukin-1. Thus the product has been shown by theresults of contacting with protease and lectins to be a non-glyco group(i.e. sugar-free) protein and by gel filtration chromatography to have amolecular weight of approximately 15,000. The biological characteristicsof the Il-1 preparation are also consistent with the definition of Il-1:

in the presence of blood serum T-cell blastogenesis is induced;

it is not able to support the growth of a permanently growing T-cellline.

In the present invention biological activity is determined by astandardized bioassay test which is carried out as follows:

Peripheral mononuclear blood cells which have been isolated andsubjected to preliminary incubation to remove serum protein andmacrophage cells adhered to the surface of the lymphocyte cells, asdescribed above, are suspended in RPMI 1640 to provide a cell density ofone million cells per ml of culture medium.

The supernatant whose biological activity is being assayed is depositedin the first row of a microtiter plate. The second row of wells isfilled with RPMI to provide a concentration which is one half that ofthe first row. Similarly, the third and each succeeding row is filledwith supernatant diluted to a concentration which is one-half of thepreceding row. As a result, a microtiter plate is obtained wherein adilution factor of 1:2 is obtained as one moves from the top towards thebottom of the plate.

A measured number of the mononuclear cells which have been preliminarilyincubated is added to each well in the microtiter plate. The growth ofthe cells as a result of stimulation by T-cell growth factor (Il-2) ismonitored. In the case of bioassay for Il-1 it is further necessary toadd serum or PHILIP to each well since otherwise Il-2 production willnot occur. Generally serum is used from a human serum pool in an amountof 15% vol/vol. Likewise, in the bioassay for the biological activity ofPHILIP either mitogen (PHA) or Interleukin-1 (serum-free) must be addedto each well containing the isolated peripheral mononuclear blood cells.

Significant activity is considered to be present in those titer rowswhere cell density has doubled at the end of seven days. The inverse ofdilution for those rows are used as the measure of significant growth.

The serum-free lectin-free Il-1 preparation so obtained, afterconcentration if desired, is used to produce Il-2 by contactinglymphocytes in the presence of PHILIP. In terms of biological activityPHILIP can be used directly after separation of the appropriatemolecular weight fraction by, for example, gel chromatography, forexample separation on a Sephadex G-150 or G-200 column (Sephadex is abead-formed, dextran gel prepared by cross-linking selected dextranfractions with epichlorhydrin; Sephadex is a trademark of Pharmacia AB,Uppsala, Sweden). Alternatively the PHILIP containing serum fraction isdialyzed against RPMI for about 24 hours using a membrane passingsubstances having molecular weights below about 10,000 to 15,000 priorto use.

However, it is preferred to use PHILIP after removal of other bloodserum proteins contained in the PHILIP-containing fraction. Since PHILIPis highly adherent to various lectins this property is utilized topurify PHILIP by affinity chromatography using the lectin as theimmobilized ligand to which the glycoprotein PHILIP is specifically (butreversibly) binding. Sepharose (a bead formed agarose gel; a trademarkedproduct of Pharmacia AB) beads have been used as the insoluble matrix towhich the ligand is immobilized. Among the ligands to which PHILIP isselectively binding are Blue A, (Cibacron Blue F3G-A, or 3G-A) and wheatgerm agglutinin (WGA). Blue A-Sepharose and WGA-Sepharose are availablefrom Pharmacia Fine Chemicals AB. Blue A is a reactive dye of thetriazinyl dye group, C.I. 61211, also known as C.I. Reactive Blue 2.Wheat germ agglutinin is the preferred lectin since other blood serumproteins of similar molecular weights, e.g. plasminogen, transferrin,and albumin are either only weakly binding or non-binding to WGA. Inparticular the following affinities of blood serum proteins to variousSapharose type affinity column gel beads can be noted:

    ______________________________________                                                  Ligand bound to Sepharose                                           Protein     ConA    WGA        Blue A                                                                              Lysine                                   ______________________________________                                        PHILIP      +       ++         ++    -                                        Plasminogen +       +          -     ++                                       Transferrin ++      +          -     -                                        Albumin     -       -          ++    -                                        ______________________________________                                         Notes:                                                                        ++ 85% of the protein is specifically bound                                   + partial specific binding                                                    - non specific binding                                                   

It can be seen from the above table that PHILIP can be easily separatedfrom other blood serum proteins in the same molecular weight range byaffinity chromatography using WGA Sepharose. The following procedure canbe used.

The biologically active fraction (capable of inducing Il-2 production inthe bioassay test) recovered from a Sephadex G200 column is incubated ona WGA Sepharose column at room temperature for about 1 hour. The columnis then eluted with two buffers. In the first elution a normal phosphatebuffer (pH=7.2) is used at a 10-fold volume (100 ml buffer)/10 ml WGASepharose). The first elution removes all proteins which are notspecifically bound by WGA.

The second elution is performed with the same phosphate buffer to whichis added N-acetyl-D-glucosamine (NADG) at about 20 mM NADG per liter ofbuffer. In the first step a 5-fold volume is used (50 ml buffer/10 mlWGA Sepharose) and this removes those proteins binding only weakly toWGA. In the second step a 5-fold volume is again used to remove allproteins with high specific binding affinity to WGA. Accordingly,essentially pure PHILIP can be obtained by the present invention.

The present invention also provides a process for producing serum-freeand mitogen and lectin free Interleukin-2 (Il-2 or TCGF) using PHILIPand the serum-free lectin-free Il-1 preparation previously described.According to this process the serum-free lectin-free Il-1 preparation ismixed with a PHILIP-containing liquid culture medium (e.g. RPMI 1640)preparation at a volumetric mixing ratio of from about 1:1 to 1:32,preferably from about 1:1 to 1:4, most preferably about 1:1.

The serum-free lectin-free Il-1 preparation can be used directly afterthe conditioning step without the Il-1 containing supernatant from thecells. In this case these cells will be activated to produce Il-2.Alternatively, the supernatant can be collected by centifugation orfiltering and a fresh supply of isolated lymphocytes provided. Is isalso possible to supply additional isolated lymphocytes to theconditioned Il-1 culture medium without removing the cells alreadycontained therein.

Although a purified PHILIP preparation can be used it is also possibleto use biologically active serum fraction from the gel chromatographyseparation. For example, if the separation is performed on a SephadexG-200 column the elution profile shows three peaks. Biological activityis found in the eluted fractions corresponding to the left shoulder ofthe third peak. Therefore, by collecting those fractions correspondingto the valley between the second and third peaks through the top of thethird peak substantially all of the biologically active T-cellregulating blood serum protein PHILIP will be recovered. These elutedfractions can then be concentrated by a factor of about 10-fold(corresponding nearly to the original concentration) by filteringthrough an appropriate filter such as Amicon YM10. The concentratedserum is then dialyzed against the sterile liquid culture medium, e.g.RPMI 1640. The amount of liquid culture medium to which the PHILIPcontaining serum fraction is added need only be that amount which givesan equivalent biological activity (as determined by the microtiterbioassay test described above) as the Il-1 preparation. However, greateror lesser amounts can be used.

This mixture is used to incubate isolated blood lymphocytes at standardtissue culture conditions: 98% humidity, 7,5% CO₂, temperature 37° C.,for about 8 to 24 hours, preferably about 8 to 18 hours.

After conditioning of the lymphocytes, specifically T-cells (presumablyT-helper cells) by the incubation, the cells are separated from theculture medium as by centrifugation for example. Some serum-free (orsubstantially serum-free, depending on the source of PHILIP) andmitogen-free T-cell growth factor (Il-2) will be present in thesupernatant since production of IL-2 can be expected to begin within afew minutes to about 1 hours after initiation of incubation.

The separated conditioned cells are repeatedly washed, for example 3times, with fresh RPMI-1640 or other suitable liquid tissue culturemedium. These cells can then be recycled for further conditioning.

The biological activity of the resulting serum-free and mitogen-freeIl-2 containing supernatant can be determined by the bioassay testdescribed above.

The liquid tissue culture medium to which pure PHILIP has been addedprovides a useful chemically defined T-cell growth culture medium whichallows for a more precise study of the regulation of T-lymphocyte growthsince the surroundings are not contaminated by other, usually undefined,proteins, as would be the case if whole serum was used. Furthermore,since human whole serum is not used the danger of transmitting hepatitisvirus is substantially eliminated.

Thus, in addition to PHILIP the liquid tissue culture medium willcontain liquid solvent, water, and conventional cell nutrients such asamino acids, vitamins, glucose, saline and a buffer system.

As described in the previously mentioned prior applications of Fabriciusand Stahn the serum-free and mitogen-free T-cell growth factor findsutility in numerous areas including, for example, diagnosis andtreatment of immune deficiency in tumor patients. Another application ofIl-2 described in the prior applications is in the field of organtransplantation. In this regard it was described that Il-2 can be usedas an antigen to develop antibodies in animals which could be used toinhibit graft rejection which is a well-known cell mediated response.

It has now been found, however, that it is possible to directlyinterfere with the ability of the immunological system to form naturalkiller cells by adding to the blood serum a serine alkylating agent.Although is is not entirely clear how the serine alkylating agentfunctions to prevent T-cell blastogenesis it is presently believed thatthe T-cell growth factor (Il-2) is a serine esterase enzyme which isinhibited from sending signals to the T-effector cells since the serineactive site is blocked by the alkylating agent. In any case, whether theIl-2 is itself a serine esterase, which is deactivated or a serineesterase is present in the serum as a comitogenic factor and this factoris prevented from cooperating with Il-2 to cause T-cell blast formation,or whether the serine alkylating agent prevents biosynthesis of Il-2 ormodifies the surface of the T-effector cell or accomplishes itsinhibitory effect in some other manner, it has nevertheless beendemonstrated in numerous experiments that the serine alkylating agentinhibits blast transformation of pre-incubated T-cells in the presenceof Il-1 and PHILIP or Il-1 and whole blood serum from humans or otheranimals, e.g. bovine animals.

Serine alkylating agents which can be used as the T-blast inhibitioninclude diisopropylfluorophosphate (DFP), phenylmethylsulfonic fluorideand the like.

It is interesting to note that plasminogen is a serine esterase.However, since it has already been found that Il-2 production and T-cellblastogenesis will take place in the absence of plasminogen it is clearthat the serine alkylating agent's reaction with plasminogen is notresponsible for the inhibition of blastogenesis.

EXAMPLE 1

Gel partition chromatography on human blood serum. Batches of 50 mlwhole human serum are applied to a Sephadex G-200 column (50×1200 mm)(from Pharmacia AB, Uppsala, Sweden) equilibrated with 0:05M phosphatebuffer containing 0,1 NaCl; flowrate 60 ml/hr temperature 4° C. and 10ml fractions were collected.

The eluate was continuously monitored for 280 nm light absorption andthe results are shown in FIG. 1. Peaks were observed at fraction numbers12, 60, 100 and 120. PHILIP activity based on the bioassay testdescribed in example 3--was found in fraction nos. 75-100. Transferrinshowed peak activity at fraction no 95. Haptoglobin showed peak activityat fraction 54. Standard immunological tests were used to measureactivity of transferrin and haptoglobin.

The results of refractionating pooled fractions 75-100 (concentrated to25 ml against polyethyleneglycol) on a Sephadex G-150 column (20×900mm); 4 ml/hr flow; 0,05M phosphate buffer containing 0:1M NaCl; is shownin FIG. 2. 3 ml fractions were collected. The different biologicalactivities can be clearly distinguished by the separated peaks.

Further differentiation of the biological activity of PHILIP andtransferrin and plasminogen is clearly demonstrated by FIG. 3 showingchromatofocussing of the pooled fractions 75-100 from the G-200 column.

EXAMPLE 2

Affinity chromatography of PHILIP

10 ml of PHILIP isolated by column chromatography as described inexample 1 is dialysed against 50 mMol/l phosphate buffer, pH 7:0 beforeapplication to affinity columns. Columns containing WGA and Blue A andConA are purchased from Pharmacia, Uppsala, Sweden. Covalent coupling of1-lysine to sepharose 4B (purchased from Pharmacia, Sweden) is performedaccording to the method described by Deutsch and Merz. (Deutsch D. G.,Mertz E. T.: (1970), Science vol. 170, pp. 1095-1096).

4 ml of PHILIP is layered into 2 ml of the affinity gels by slowaddition, drop by drop. The column is incubated at room temperature for60 minutes whereafter the non-adsorbed material is eluted by a 50 mMol/lphosphate buffer, pH 7:0. A buffer volume of 50 ml is used. Then theadsorbed material is eluted by use of a 50 mMol/l phosphate buffer, pH7:0 containing N-acetyl D glucosamine in a concentration of 200 mMol/lin the case of WGA, alpha-D-mannopyranoside 200 mMol/l in the case ofConA, epsilon-amino capronic acid 200 mMol/l in the case of lysine andNADPH in the same concentration dissolved in the same buffer containingadditionally 50% ethylene glycol and 20% NaCl in the case of Blue A. Theeluted samples--for each column two 50 ml portions are eluted--areconcentrated by polyethylene glycol to an end volume of 2 ml anddialysed against RPMI exhaustively. PHILIP is recovered fromWGA-sepharose in the second specific eluate.

EXAMPLE 3

Biological test for PHILIP activity

10 ml of the collected fraction from the column chromatography inexample 1 is dialyzed against 200 ml of RPMI 1640. The dialysis isperformed at 4° C. Thereafter the preparation is sterilized byfiltration through a 0:22 um sterile filter which is available fromdifferent commercial sources and pipetted into microtiter plates whichhave been prefilled with RPMI 1640 to a volume of 200 ul/ well, so thatin the first well of a row 200 μl PHILIP is mixed to the preexistingRPMI. After mixing the volume of 200 ul is transferred to the secondwell of the row and so on, until a dilution of 1:32 has been achieved50,000 cells which have been incubated at 37° C., 98% humidity and 10%CO₂ in the serum-free RPMI for 24 hours are added in a volume of 20-50ul to each well in plate. PHA is added to a final concentration of 4ug/ml to each well. The microtiter plate is incubated for 7 days underthe conditions described above. On day 7 the plate is assayed for cellgrowth by counting the cells in an electronic cell counter. The dilutionof the PHILIP preparation in which the cell number has doubled (i.e.reached 100,000) is considered as the inverse of the titer. If forexample the cell number has increased to more than 100,000 in thedilution of 1:8, the titer in the preparation in considered to be 8.

EXAMPLE 4

Inactivation of PHILIP by trypsin and by neuraminidase enzymes.

a) Inactivation by trypsin 10 ml samples of PHILIP obtained by columnchromatography as described in example 1 are incubated at 37° C. inpresence of trypsin (4x crystallized, obtained e.g. from SERVA ChemicalCorp. Heidelberg, FRG) concentration 0:1% w/v in phosphate buffer, pH7:0 in the presence of 50 mMol/l CaC12. After 120 min. incubation, EDTAis added in a concentration of 0:01 mMol/l whereafter the samples areincubated again at 37° C. for 120 min. After this second incubation 10mMol/l DFP is added to the sample in order to inhibit the trypsin whichis a serine protease.

The sample is dialysed against 200 ml RPMI with a dialyzing membranepermeable to substances having molecular weight below about 15,000 andtested for biologic activity according to the test method described inexample 3. No biological activity was observed.

b) Inactivation by neuraminidase

Neuraminidase isolated from Clostridium botulinus which is readilycommercially available is covalently coupled to BrCN-activated Sepharose4B manufactured by Pharmacia, Uppsala, Sweden, whereafter the gel isthoroughly rinsed in Phosphate buffer. PHILIP samples as described inpart a) of this example, are layered onto the top of columns with a gelcontent of 2 ml and incubated at 37° C. for 6 hours under sterileconditions. Thereafter the sample is eluted by phosphate buffer, pH 7:0and dialysed against RPMI 1640 as described above. The biologic activityof PHILIP is determined according to the microtiter bioassay techniquedescribed above in example 3. No biological activity was observed.

EXAMPLES 5

Production of an Interleukin-1 preparation free of mitogen and serumproteins

a) Preparation by use of adherent pro-macrophages Blood lymphocytes areisolated by the following procedure which is described in example 1 ofthe above mentioned application Ser. No. 193,112, now abandoned andwhich is identical to Example 1 of U.S. Pat. No. 4,390,623.

Human blood contained in a one-pint plastic bag and mixed with aclotting inhibitor, heparin, is obtained from a blood bank. Twenty 50 mlsterile tubes are prefilled with Ficoll-hypaque mixture. The blood islayered into the Ficoll-hypaque containing tubes. The tubes arecentrifuged at 400G for ten minutes. As a result of the centrifugation,a ring of the peripheral mononuclear blood cells is formed above theFicoll-hypaque layer in each tube. This layer of the peripheralmononuclear blood cells is drawn off carefully by means of a pipet. Thecells collected from ten tubes are deposited into one 50 ml tube, theamount of the cells added being approximately 25 ml. To this tube isadded about 25 ml of RPMI 1640 solution in order to dilute theconcentration of the Ficoll-hypaque in the tube. Since Ficoll-hypaque isslightly toxic, it should be separated from the cells carefully.

The tube containing the cells and RPMI 1640 is centrifuged for tenminutes at 300G. The centifugation forces are kept low in order to avoidunnecessary breakage of the cells. After centrifugation, the supernatantis discarded.

A fresh portion of RPMI solution is added to the tube. The cells arewashed by resuspending the cells in the solution. Such resuspension isconducted by drawing the solution and cells into a pipet and thenpermitting the mixture to flow back into the tube. Once again, thesuspension of the cells is conducted in a gentle manner in order toavoid breakage of the cells. The suspension is then centrifuged for tenminutes at 300G to obtain a cell pellet and a supernatant which isdiscarded. This washing procedure is repeated for a total of threetimes, with the supernatant obtained in each washing being discarded anda cell pellet being retained. After washing, the cells are suspended infive to ten RPMI 1640 solution and counted. After washing with sterileculture medium, the cells, usually 400 to 800 millions isolated per 400ml whole blood , are incubated 60 minutes at 37° C. in RPMI 1640 understandard cell culture conditions. The incubation is performed on aplastic petri dish with a diameter of 15 cm. Such dishes arecommercially readily available. After incubation, the cell containingmedium is poured off the dish, and the dish with the adhering cells,usually 10% of the total cell number is rinsed several times, usually 6,with RPMI 1640 to remove all non-adherent cells. Thereafter the adherentcells are removed from the dish by use of a rubber policeman, countedand suspended in a concentration of 3 millions/ml in RPMI 1640 andincubated 24 hours at 37° C. under standard cell culture conditionsbefore further processing. After incubation the cells are stimulatedwith PHA for 4 to 12 hours preferably 4 to 8 hours, most preferably 4hours at a concentration of 4 μg PHA/ml of culture medium. The PHA isthen discarded, the cells washed 3 times as described above, whereafterthe cells are incubated in RPMI 1640 without supplements for 18-24hours, preferably 20 hours. The culture supernatants are collectedwhereafter the cells can be stimulated again with PHA if so desired. Thestimulation may be repeated up to 3 times if so desired. The serum freeculture supernatants can be concentrated on a YM 10 membrane asdescribed in example 1 of Ser. No. 193,112, now abandoned and which isidentical to example 1 of U.S. Pat. No. 4,390,623, if desired. The IL-1containing supernatants can be stored at -20° until they are used.

b) Preparation by use of isolated blood lymphocytes without separationof adherent macrophages.

Blood lymphocytes are isolated as described above in this example. Thecells after washing with RPMI 1640 are incubated at 37° C. understandard cell culture conditions for 24 hours. Thereafter the cells arecounted and stimulated with PHA at a concentration of 4 μg/ml culturemedium and a cell density of 3 millions/ml as described above in thisexample. All remaining steps of the preparation of IL-1 are the same asthose mentioned above in this example.

EXAMPLE 6

Biological test for Interleukin-1 activity

Interleukin-1, free of lectin and sera, prepared as described in example5 is filtered sterile and tested for its biological activity as follows:RPMI 1640 is mixed with either 15% inactivated human AB serum or withPHILIP to a titer of 8 in the end volume. 200 μl thereof is introducedinto the top row of wells in a microtiter plate. In the following rows100 ml of the mixture is applicated to each well. 200 μl of the Il-1 isadded to the wells of the top row and mixed with the RPMI 1640suplemented by serum PHILIP. 100 μl is transferred to the next row,mixed and so on. Thus a serial dilution of Il-1 in dilution steps of 1:2is achieved. 20 to 50 μl of isolated blood lymphocytes as described inexample 1 of application Ser. No. 193,112, now abandoned and which isidentical to Example 1 of U.S. Pat. No. 4,390,623 are added. As aconsequence of the interaction of Il-1 and PHILIP, the cells starsynthesizing Il-2. The effectiveness of the synthesis is dependent onthe concentration of Il-1. Il-1 activity is quantificated by the titerit exhibits in the culture (i.e. by the inverse of the dilution in whichthe cell number has doubled within seven days in culture).

EXAMPLE 7

Production of Interleukin-2 by use of PHILIP

a) Production of Interleukin-2 by use of PHILIP and PHA Peripheral bloodlymphocytes isolated as described above, after washing 3 × with RPMI1640, are suspended in a mixture of RPMI 1640 and PHILIP isolated asdescribed above in example 1. PHILIP is present at a concentration of1:1 and 1:32, usually 1:16. The concentration of PHILIP is determined byits biological activity, which is assayed according to example 3. Atiter of 8 in the end volume is desired. The cells are suspended at aconcentration of 3 millions/ml and stimulated 4 to 24 hours with PHA ina concentration of 4 μg/ml. The stimulation time is preferably 8-20hours, most preferably 8-12 hours. After stimulation, the cells arewashed 3 times as described above, and used to condition the culturemedium at a concentration of 3 millions/ml in absence of othersupplements than those contained in RPMI 1640. The conditioning timeshould be 18- 24 hours, most preferably 20 hours. Thereafter the cellsare spun down and the culture medium collected. The stimulation stepmight be repeated up to 3 times, if so desired. The conditioned mediumcan, if desired, be concentrated on a YM 10 membrane as described above.Interleukin-2 activity can be assayed by either one of the twobiological tests described above in this application. The activity ofIL-2 will rise as a direct function of the degree of concentration. Themedium containing PHA and PHILIP can be re-used up to 3 times forgeneration of Il-2.

b) Production of Interleukin-2 by use of PHILIP and Interleukin-1

Mitogen- and serum-free IL-1 preparations as described above in thisapplication are mixed with PHILIP isolated according to the methoddescribed above in this application in such a proportion that PHILIPwill be present in a final titer of 8. The so prepared culture medium isused under the same conditions as is PHA and PHILIP containing culturemedium used in this example. The processing of the IL-2 preparation isthe same as described above in this example.

EXAMPLE 8

Inhibition of blast transformation and of continuous Il-2 dependentT-cell growth by serine esterase inhibitors.

Cells used for blast formation assay are cells which according to themethod described above have been incubated 24 hours in serum free RPMIfor 24 hours. Cells used for testing the Il-2 dependent T-cell growthare derived from a continuously growing T-cell line kept in culture inour labor 24 hours. Cells used for testing the Il-2 dependent T-cellgrowth are derived from a continuously growing T-cell line kept inculture in our laboratory. The preparations to be inactivated by serineesterase inhibitors are incubated either with DFP(diisopropylfluorophosphate) in a concentration of 0:0024 ml per mlpreparation (usually 5 ml preparation to be tested is mixed with 60 ulof DFP), or with PMSF (phenylmethylsulfonylfluoride) in a concentrationof 50 mMol/l of preparation to be tested. The inactivation is performedat 0° C. After 60 minutes, the preparation is transferred into dialysisbags and dialysed against 1000 ml of RPMI for 6 hours. Then the RPMI ischanged against the same volume of fresh RPMI and dialysis is continuedfor 24 hours at 4° C. The biological test is performed in the followingway:

The inactivated preparations are transferred to microtiter plates afterthey have been sterilized by filtration as described in example 4. Cellsare added to the wells at a concentration of 50,000/well, and the cellnumber is counted on day 7 as described in example 4. Cell counts lessthan 100,000 are considered indicative of loss of Il-2 activity.

In our experiments we have always either yielded cell counts either ofmore than 100,000 or less than 50,000 which was the cell numberintroduced to the well. Usually a positive test yields cell counts of400,000 to 600,000.

What is claimed is:
 1. A human Interleukin-1 preparation produced by aprocess comprising(a) incubating isolated human peripheral mononuclearblood cells comprising at least Interleukin-1 producing monocytic cellsin a serum-free liquid tissue culture medium to remove serum proteinsadhering to the cell membranes; (b) contacting the incubated blood cellswith a mitogen for a period of about 4 to 12 hours to activate themonocytic cell population of the mononuclear blood cells for theproduction of Interleukin-1; (c) washing the activated cells with asterile liquid to remove the mitogen; and (d) incubating the activatedcells in a serum-free liquid tissue culture medium to collectInterleukin-1 in the liquid phase of the tissue culture medium tothereby obtain a preparation of Interleukin-1.
 2. The humanInterleukin-1 preparation of claim 1 wherein the human peripheralmononuclear blood cells in step (a) are incubated for about 18-24 hours.3. The human Interleukin-1 preparation of claim 1 wherein the humanperipheral mononuclear blood cells used in step (a) are isolatedpromacrophage cells separated from peripheral mononuclear blood cells.4. The human Interleukin-1 preparation of claim 1 wherein the mitogenused in step (b) is phytohemagglutinin A and the incubated cells areleft in contact with the mitogen for about 4 hours.
 5. The humanInterleukin-1 preparation of claim 1 wherein the activated cells in step(d) are incubated for about 18 to about 24 hours.